MicroRNAs (miRNAs, miRs) are found in over one hundred distinct organisms, including fruit flies, nematodes and humans. miRNAs are believed to be involved in a variety of processes that modulate development in these organisms. The miRNAs are typically processed from 60- to 70-nucleotide foldback RNA precursor structures, which are transcribed from the miRNA gene. The RNA precursor or processed miRNA products are easily detected, and a lack of these molecules can indicate a deletion or loss of function of the corresponding miRNA gene.
Cancers are a significant source of mortality and morbidity in the U.S. and throughout the world. In particular, chronic lymphocytic leukemia (“CLL”) and other BCL2-associated cancers (e.g., acute myeloid leukemia, multiple myeloma, melanomas, lymphomas (e.g., follicular lymphoma, large cell lymphoma, non-Hodgkin's lymphoma), carcinomas (e.g., brain carcinoma, breast carcinoma, prostate carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocellular carcinoma and gastric carcinoma), hematologic malignancies, solid tumors, colorectal cancer, Epstein-Barr virus-associated lymphoproliferative disease) are clinically important neoplastic diseases of adult humans. For example, CLL is the most common form of adult leukemia in the Western world, and the age-adjusted incidence of prostate cancer now surpasses that of all other cancers among men in the United States, and, after lung cancer, is the second leading cause of all male cancer deaths in the country.
Hemizygous and/or homozygous loss at 13q14 occurs in more than half of the reported CLL cases, and constitutes the most frequent chromosomal abnormality in CLL. The karyotyping of tissue samples from CLL patients identified relatively few chromosomal abnormalities, suggesting that the specificity and frequency of observed deletions at 13q14 have pathologic significance. In addition, 13q14 deletions also occur in 60% of prostate cancers, suggesting that one or more tumor suppressor genes located at 13q14 are involved in the pathogenesis of both CLL and prostate cancers.
The presence of both clonal homozygous and heterozygous deletions, and the very high frequency of 13q14 loss in CLL and prostate cancers, indicates that deletions in this region are related to the etiology of certain cancer types. Several groups have used positional cloning in order to identify the gene or genes in the deleted areas. To date, a total of eight genes from the deleted regions of 13q14 in sporadic and familial cases of CLL have been identified and screened for alterations at the DNA and/or RNA level: Leu1 (BCMS or EST70/Leu1), Leu 2 (ALT1 or 1B4/Leu2), Leu 5 (CAR), CLLD6, KPNA3, CLLD7, LOC51131 (putative zinc finger protein NY-REN-34 antigen) and CLLD8. However, detailed genetic analyses, including extensive loss of heterozygosity (LOH), mutation and expression studies, have failed to demonstrate the consistent involvement of any of these genes in carcinogenesis.
The malignant, mostly non-dividing B cells of CLL overexpress Bcl2 protein (Kitada, S., et al., Blood 91: 3379-3389 (1998)), an apoptosis inhibitor that plays a central role in promoting survival of eukaryotic cells by inhibiting cell death (Cory, S., and Adams, J. M., Nature Reviews 2: 647-656 (2002)). Overexpression of Bcl2 has been associated with many types of human cancers, including leukemias, lymphomas and carcinomas (Sanchez-Beato, M., et al., Blood 101: 1220-1235 (2003)). In follicular lymphomas, and in a fraction of diffuse B-cell lymphomas, the mechanism of BCL2 activation was found to be a chromosomal translocation, t(14;18)(q32;q21), which places the BCL2 gene under the control of immunoglobulin heavy chain enhancers, resulting in deregulated expression of the gene (Tsujimoto, Y., et al., Science 226: 1097-1099 (1984); Tsujimoto, Y., et al., Science 228: 1440-1443 (1985)). However, the BCL2 gene is juxtaposed to immunoglobulin loci in less than 5% of CLL cases (Adachi, M., et al., J. Exp. Med. 171: 559-564 (1990)), and the mechanism by which BCL2 is overexpressed in the majority of CLL cancers is unknown.
Current therapies for CLL typically involve chemotherapy, administered alone or in combination with autologous bone marrow transplantation. The chemotherapy agents employed are generally toxic to the patient and cause only partial remissions in a relatively large proportion of patients. Therapies for BCL2-associated cancer therapies can also involve chemotherapy, often following surgical resection of a tumor. However, as with CLL, the curative properties of the chemotherapeutic agents (with or without surgery) are limited.
Treatment with chemotherapy alone is limited in that cancer cells often become resistant to a broad spectrum of structurally unrelated chemotherapeutic agents. Such resistance, termed “multidrug resistance” (MDR), is a common problem in the treatment of patients with cancer, and the resistance of tumor cells to chemotherapeutic drugs represents a major problem in clinical oncology.
Apoptosis is an important component of the sequence of events during which chemotherapeutic drugs induce an antitumor response, and studies have implicated Bcl2 as having a critical role in anticancer drug-induced apoptosis (Kim, R et al., Cancer 101(11): 2491-2502 (2004)). Furthermore, tumor cells engineered to overexpress Bcl2 develop resistance to the cytotoxic effects of a number of different drugs (Kamesaki et al., Cancer Res. 53: 4251-4256 (1993); Miyashita and Reed, Blood 81: 151-157 (1993)).
There is a need for a rapid, economical and accurate diagnostic test for CLL and other BCL2-associated cancers. There is also a need for an economical and effective treatment for BCL2-associated cancers, such as CLL, which does not have a significant negative impact on the patient. There is a further need for new anti-cancer therapies that are directed to inhibiting expression of Bcl2, given that a wide variety of common cancers are associated with Bcl2 protein overexpression. In addition, there is a need for improved anti-cancer therapies that display increased efficacy relative to other conventional anti-cancer treatments. Furthermore, there is a need to identify agents and methods that can increase the sensitivity of cancer cells to the cytotoxic effects of anti-cancer agents, thereby improving cancer therapies. There is also a need for methods of inducing apoptosis in a cell that overexpresses an anti-apoptotic protein, such as Bcl2.